Production of chimeric bovine or porcine animals using cultured inner cell mass

a technology of inner cell mass and which is applied in the field of production of chimeric bovine or porcine animals using cultured inner cell mass, can solve the problems of insufficient cell culture, inability to produce transgenic ungulate embryos produced from transgenic es cells, and inability to manipulate or otherwise control dna integration

Inactive Publication Date: 2006-01-26
UNIV OF MASSACHUSETTS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0038] It is another object of the invention to provide novel methods for the isolation and / or production of cultured ungulate ICM cells or cell lines which exhibit morphological characteristics and which express cell markers identically or substantially similarly to the ICM of developing ungulate embryos, preferably for long periods in culture.

Problems solved by technology

However, the direct injection of DNA into pronuclei does not provide an opportunity to manipulate or otherwise control DNA integration.
However, to date, the production of transgenic ungulate embryos produced from transgenic ES cells has not been reported.
Also, these cells are not cultured under conditions wherein they maintain constant contact with a fibroblast feeder layer.

Method used

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  • Production of chimeric bovine or porcine animals using cultured inner cell mass
  • Production of chimeric bovine or porcine animals using cultured inner cell mass
  • Production of chimeric bovine or porcine animals using cultured inner cell mass

Examples

Experimental program
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Effect test

example 1

[0177] Production of CICM cell lines from pre-blastocyst and blastocyst stage pig embryos were conducted using the following general protocol. First, primary cultures of embryonic fibroblasts were obtained from 12-16 day old murine fetuses. After the head, liver, heart and alimentary tract were aseptically removed, the embryos were minced and incubated for 30 minutes at 37° C. in prewarmed trypsin EDTA solution (p. 05% trypsin / 0.02% EDTA; GIBCO, Grand Island, N.Y.). Fibroblast cells were plated in tissue culture dishes and cultured in alpha-MEM medium (BioWhittaker, Walkersville, Md.) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, Utah) penicillin (100 IU / ml) and streptomycin (50 μg / ml). Three to four days after passage, embryonic fibroblasts, in 35×10 Nunc culture dishes (Baxter Scientific, McGaw Park, Ill.), were treated with mitomycin C (10 μg / ml; Sigma) in supplemented alpha MEM for a minimum of three hrs. The fibroblasts were grown and maintained in a humidified ...

example 2

[0184] CICM cells obtained according to Example 1 were used for insertion of heterologous DNA's. Specifically, these cells were microinjected with linear as well as supercoiled DNA constructs containing different promoters placed in front of either the beta-galactosidase gene and / or the neomycin phosphotransferase gene. The specific promoters used were the cytomegalovirus promoter (CMV promoter), phosphoglycerate Kinase promoter (PGK promoter), mammary promoter (MAM promoter), reCMV promoter and chicken beta actin promoter. These gene constructs were diluted in a buffered solution (containing 80 mM KCl and 70 mM HEPES.) However, other buffers may readily be substituted therefore, such as Tris EDTA. The concentration of the DNA constructs in solution ranged from 5 to 10 μg / ml. However, concentrations ranging from 0.1 to 100 μg / ml should be effective. These DNA preparations were then microinjected into cultured CICM cells obtained according to Example 1.

[0185] In this procedure, the ...

example 3

[0193] CICM cells obtained from cattle according to Example 1 were used for insertion of heterologous DNAs followed by selection in vitro for cells that stably expressed the heterologous DNAS. In this example the cytomegalovirus promoter and a fusion gene consisting of both the beta-galactosidase gene and the neomycin phosphotransferase gene (beta-GEO) were used. Microinjection of the gene construct into the CICM cells followed the same procedure as described in Example 2 for both the bovine and the porcine CICM cells. The CICM cells were propagated on primary mouse embryonic fibroblast cells derived from a line of transgenic mice (commercially available from Jackson Labs) that have the neomycin phosphotransferase gene in their genome. The day after the CICM cells were microinjected they were placed in selection growth medium containing the selection compound G418 (genticin). This drug kills any cell that is not expressing the neomycin phosphotransferase gene.

[0194] The transgenic ...

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Abstract

Novel cultured inner cell mass (CICM) cells, and cell lines, derived from ungulates, in particular, pigs and cows, and methods for their preparation are provided. The subject CICMs possess similar morphology and express cell markers identically or substantially similarly to ICMs of undifferentiated developing embryos for prolonged culturing periods. Heterologous DNA is inserted into the subject CICM cells and cell lines so as produce transgenic CICM cell which are introduced into non-human fertilized embryos to produce transgenic chimeric embryos. The transgenic chimeric embryos are transferred into recipient females where they are permitted to develop into transgenic chimeric fetuses. Recipient females give birth to transgenic chimeric animals which are capable of transmitting the heterologous DNA to their progeny. Transgenic CICM cells are also used to produce cloned transgenic embryos, fetuses and offspring.

Description

RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. application Ser. No. 08 / 626,054.FIELD OF THE INVENTION [0002] Novel cultured inner cell mass (CICM) cells, cell lines, and methods for their preparation are provided. The subject CICMs possess similar morphology and express cell markers identically or highly similarly to ICMs of developing embryos for prolonged culturing periods. These CICMs are produced by novel culturing techniques and / or by introduction of a regulatable differentiation inhibiting gene (DI). The subject CICM cell lines are used to produce differentiated cells, tissues, organs and / or whole animals, preferably ungulates, desirably those which have been genetically modified to contain within their genome a desired heterologous DNA or which have been selected to contain genetically desirable traits. This is accomplished by in vitro or in vivo culture techniques or by producing chimeric or nuclear transfer embryos, fetuses and / or ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027
CPCA01K2217/05C12N15/8778C12N15/8771
Inventor STICE, STEVEN L.CIBELLI, JOSEROBL, JAMESGOLUEKE, PAULLEON, F. ABEL PONCE DEJERRY, D. JOSEPH
Owner UNIV OF MASSACHUSETTS
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